Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice

Background Extracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA. Materials and Methods To determine whether Dspp+/- mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/-, and Dspp homozygous (Dspp-/-) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/- mice over time, condyles of Dspp+/- and WT mice were analyzed radiologically, histologically, and immunohistochemically. Results Micro-CT and histomorphometric analyses revealed that Dspp+/- and Dspp-/- mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp-/- mice which exhibited tooth loss, Dspp+/- mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/- mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/- mice showed increased distribution of osteoclast in the subchondal bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage. Conclusions Dspp+/- mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers. Overall design: To investigate the haploid deletion of DSPP affects the biological processes of ossification and osteoclast differentiation, We obtained the mandibular condyles from 4-month DSPP+/-, -/-, and wild-type mice.We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different tissues.Comparative gene expression profiling analysis of RNA-seq data for Osteoblast/osteoclast differentiation markers.

细胞外基质（Extracellular matrix, ECM）蛋白功能异常或缺陷可引发颞下颌关节骨关节炎（temporomandibular joint osteoarthritis, TMJ OA）。牙本质涎磷蛋白（dentin sialophophoprotein, DSPP）属于下颌髁突软骨的细胞外基质蛋白，其缺失会影响出生后髁突软骨的细胞增殖及其他细胞外基质相关改变。目前，DSPP长期功能缺失是否会导致颞下颌关节骨关节炎仍不明确。本研究旨在验证以下假说：DSPP长期单倍体不足会引发颞下颌关节骨关节炎。

材料与方法 为明确Dspp+/-小鼠是否可在无严重牙齿缺损的情况下表现出颞下颌关节骨关节炎，研究采用显微计算机断层扫描（Micro-computed tomography, micro-CT）对野生型（wild-type, WT）、Dspp+/-及Dspp纯合型（Dspp-/-）小鼠的下颌骨进行分析。为表征Dspp+/-小鼠随时间推移的骨关节炎退变进程与潜在机制，研究通过放射学、组织学及免疫组织化学方法对Dspp+/-与野生型小鼠的髁突进行检测分析。

结果 显微CT与组织形态计量学分析显示，与野生型小鼠相比，12月龄的Dspp+/-及Dspp-/-小鼠的软骨下骨质量、骨体积分数、骨密度及骨小梁厚度均显著降低。值得注意的是，与出现牙齿脱落的Dspp-/-小鼠不同，Dspp+/-小鼠仅存在轻微牙齿缺损。RNA测序（RNA-seq）数据表明，DSPP单倍体不足会影响骨化与破骨细胞分化的生物学过程。此外，组织学分析显示，Dspp+/-小鼠存在髁突软骨裂隙、软骨厚度降低、关节细胞数量减少及严重的软骨下骨空腔，且上述表现随年龄增长而进一步加重。放射学数据显示，至18月龄时小鼠软骨下骨质疏松程度加剧，21月龄时出现骨赘形成。同时，Dspp+/-小鼠的软骨下骨中破骨细胞分布增加，下颌髁突软骨内的基质金属蛋白酶2（MMP2）、白细胞介素6（IL-6）、纤连蛋白1（FN-1）及Toll样受体4（TLR4）表达水平均升高。

结论 Dspp+/-小鼠会以时间依赖性方式出现颞下颌关节骨关节炎，其下颌髁突病变源于软骨下骨矿化不足与下颌髁突软骨的破坏，同时伴随炎症标志物的上调表达。

整体实验设计 为探究DSPP单倍体缺失对骨化与破骨细胞分化生物学过程的影响，本研究从4月龄的DSPP+/-、DSPP-/-及野生型小鼠体内获取下颌髁突。随后基于3种不同组织的RNA-seq数据开展基因表达谱分析，并对成骨细胞/破骨细胞分化标志物的RNA-seq数据进行比较基因表达谱分析。